JPWO2018021046A1 - Transformant producing 3HH unit-containing copolymerized PHA, and method for producing the PHA - Google Patents

Abstract

Provided are a transformant producing a copolymerized PHA containing 3HH units at a higher composition ratio, and a method of producing a copolymerized PHA using the transformant. For a prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing 3HH units, an enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity is encoded A transformant producing a co-polymerized PHA containing 3HH units, into which a gene for A method for producing a copolymerized PHA containing 3HH units, comprising the step of culturing the transformant.

Description

  The present invention relates to a transformant producing a copolymerized PHA containing 3HH units, and a method for producing the PHA.

  Polyhydroxyalkanoate (hereinafter sometimes referred to as PHA) is a polyester type organic polymer produced by a wide range of microorganisms. PHA is a biodegradable thermoplastic polymer and can produce renewable resources as a raw material. For these reasons, attempts have been made to industrially produce PHA as an environmentally friendly material or a biocompatible material and to use it in various industries.

  To date, many microbes are known to accumulate PHA in cells as an energy storage material. Representative examples of PHA include poly-3-hydroxybutyric acid (hereinafter also referred to as P (3HB)), which is a homopolymer of 3-hydroxybutyric acid (hereinafter also referred to as 3HB). P (3HB) is a thermoplastic polymer, and is biodegradable as it is in the natural environment. However, P (3HB) is hard and brittle due to its high crystallinity, and its application range is practically limited. In order to expand the application range, it was necessary to give P (3HB) flexibility.

  Therefore, a copolymerized PHA (hereinafter referred to as P (3HB-co-3HV) consisting of 3HB and 3-hydroxyvaleric acid (hereinafter referred to as 3HV) and a method for producing the same have been developed (for example, Patent Document 1 and See Patent Document 2). P (3HB-co-3HV) is considered to be applicable to a wide range of applications because it is more flexible than P (3HB). However, in fact, even if the 3HV mole fraction in P (3HB-co-3HV) is increased, the accompanying change in physical properties is scarce, and it is particularly required for processing into films, sheets, flexible packaging containers, etc. Because the flexibility is not improved to such an extent, it is used only in the limited field of hard molded articles such as shampoo bottles and disposable razor handles.

  Furthermore, in order to increase the flexibility of PHA, a copolymerized PHA (hereinafter sometimes referred to as P (3HB-co-3HH)) composed of 3HB and 3-hydroxyhexanoic acid (hereinafter sometimes referred to as 3HH), and Research has been conducted on the manufacturing method (see Patent Document 3 and Patent Document 4). In these reports, P (3HB-co-3HH) is produced by fermentation using a wild strain of Aeromonas caviae isolated from soil and a fatty acid such as oleic acid or palmitic acid as a carbon source .

  Studies on physical properties of P (3HB-co-3HH) have also been conducted (see Non-Patent Document 1). In this report, fatty acids having 12 or more carbon atoms are the only carbon source. caviae is cultured to ferment and produce P (3HB-co-3HH) having various 3HH composition ratios. As P (3HB-co-3HH) increases in the 3HH compositional ratio, it becomes more flexible from hard and brittle properties such as P (3HB), and becomes higher as the 3HH compositional ratio becomes higher. It has been shown to exhibit flexibility over co-3 HV). That is, since P (3HB-co-3HH) can have wide physical properties applicable from hard polymers to soft polymers by changing the 3HH composition ratio, application to a wide range of fields can be expected.

  In addition, the plasmid pJRD215 (ATCC 37533) is a plasmid derived from a plasmid PHA synthetase expression plasmid such as pJRDEE32 or pJRDEE32d13, in which a polyester synthetase gene or an R-body specific enoyl CoA hydratase gene is introduced into the plasmid pJRD215 (ATCC 37533). The PHA productivity of the transformed transformant has been investigated (see Patent Document 5 and Non-patent Document 2). Although the amount of cells after cultivation of the strain was originally as low as 4 g / L, the amount of cells was up to 45 g / L and the polymer content was up to 62.5% by improving the culture conditions of the strain using plant oil as a carbon source. It was found that the polymer productivity was improved and the 3HH composition ratio was improved to 8.1 mol%. Thus, attempts have been made to improve the 3HH composition ratio and polymer productivity of P (3HB-co-3HH) depending on culture conditions (see Patent Document 6).

  R-body specific enoyl CoA hydratase gene is There is also a report that the 3HH composition ratio is improved by incorporating it into the chromosomal DNA of necator (see Patent Document 7 and Non-patent Document 3). In this report, C.I. By inserting multiple R-body specific enoyl CoA hydratase genes into the pha operon region including the pha synthetase gene of necator, the 3HH composition ratio of PHBH produced using plant oil as a raw material is improved to 10.5 mol% .

  Like these, production of P (3HB-co-3HH) is often performed using vegetable oil and fat as a raw material. On the other hand, research has also been conducted to produce P (3HB-co-3HH) from carbohydrate raw materials (see Non-Patent Document 4). In this report, C.I. By introducing a crotonyl-CoA reductase gene derived from Streptomyces cinnamomonensis into necator, P (3HB-co-3HH) containing a small amount of 3HH units is produced from fructose as a raw material.

  Also, in recent years, it has been reported that the 3HH composition ratio is improved by introducing an ethylmalonyl-CoA decarboxylase gene in addition to the crotonyl-CoA reductase gene (see Non-Patent Document 5).

Japanese Patent Application Laid-Open No. 57-150393 Japanese Patent Application Laid-Open No. 59-220192 Unexamined-Japanese-Patent No. 5-93049 Unexamined-Japanese-Patent No. 7-265065 gazette JP 10-108682 A Japanese Patent Application Publication No. 2001-340078 International Publication No. 2011/105379

Y. Doi, S. Kitamura, H. et al. Abe, Macromolecules, 28, pp. 4822-4823 (1995) T. Fukui, Y. Doi, J. et al. Bacteriol, 179, 15, pp. 4821-4830 (1997). Y. Kawashima et al. , Appl. Environ. Microbiol. , 78, pp. 493-502 (2012) T. Fukui, H. Abe, Y .; Doi, Biomacromolecules, 3, pp. 618-624 (2002). C. Insomphun, H. et al. Xie, J., et al. Mifune, Y .; Kawashima, I. Orita, S. Nakamura, T .; Fukui, metabolic engineering, 27, pp. 38-45 (2015)

  An object of the present invention is to provide a transformant that produces a copolymerized PHA containing 3HH units at a higher composition ratio, and a method for producing a copolymerized PHA using the transformant.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors found that trans-2-enoyl-CoA hydratase activity, which is usually not retained in prokaryotes and retained only in eukaryotes, and (R)- By introducing a gene encoding an enzyme having both 3-hydroxyacyl-CoA dehydrogenase activity into a prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing 3HH unit, the 3HH unit is higher It has been found that the fermentative production of copolymerized PHA contained in the composition ratio is possible, and the present invention has been completed.

  That is, the present invention provides trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase for a prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing a 3HH unit. The present invention relates to a transformant producing a co-polymerized PHA containing 3HH unit into which a gene encoding an enzyme having activity is introduced.

  The transformant is preferably further introduced with a gene encoding crotonyl-CoA reductase (CCR), and further, a gene encoding ethylmalonyl-CoA decarboxylase is introduced. Is preferred.

  The gene encoding the enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity is a gene derived from Yarrowia lipolytica or a gene derived from Drosophila melanogaster Is preferred.

  The prokaryotic microorganism is preferably a bacterium, more preferably a bacterium belonging to the genus Capriavidus, and still more preferably a compound of Capriabidus nectar.

  The second present invention relates to a method for producing a copolymerized PHA containing 3HH unit, which comprises the step of culturing the transformant. The copolymerization PHA is preferably P (3HB-co-3HH).

  According to the present invention, it is possible to provide a transformant producing a copolymerized PHA containing 3HH units at a higher composition ratio. Further, by culturing the transformant, it becomes possible to fermentatively produce a copolymerized PHA containing 3HH units at a higher composition ratio.

  Hereinafter, the present invention will be described in more detail.

In the present invention, trans-2-enoyl-CoA hydratase activity is applied to a prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing 3HH units. By introducing a gene encoding an enzyme having the (R) and (R) -3-hydroxyacyl-CoA dehydrogenase activity, a transformant producing a copolymerized PHA containing a 3HH unit at a higher composition ratio is provided.

  In the present invention, the prokaryotic microorganism as the original strain into which the gene encoding the enzyme is introduced is not particularly limited as long as it is a prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing 3HH units. As such a prokaryotic microorganism, not only a wild strain inherently having the PHA synthetase gene, but also a mutant obtained by artificially mutating such a wild strain, or an exogenous gene by genetic engineering techniques Or a recombinant prokaryotic microorganism strain into which a PHA synthetase gene and / or a PHA production-related enzyme gene has been introduced.

  The possibility of synthesizing a copolymerized PHA in which the PHA synthetase gene contains a 3HH unit does not mean that a copolymerized PHA containing a 3HH unit can be synthesized under any culture conditions, but a specific culture condition It is sufficient if the copolymerized PHA containing 3HH units can be synthesized. For example, the strain described in Comparative Example 2 described later does not synthesize a copolymerized PHA containing 3HH units under culture conditions using glucose as a single carbon source, but under culture conditions containing oil and fat as a carbon source, 3HH units The present invention corresponds to “prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing a 3HH unit” since it can synthesize a copolymerized PHA containing

  Specific examples of such prokaryotic microorganisms include bacteria, actinomycetes, cyanobacteria, archaea and the like, with preference given to bacteria (bacteria). Examples of the bacteria include, for example, Ralstonia genera, Cupriavidus genera, Wautersia genera, Aeromonas genera, Aeromonas genera, Escherichia genera, Alcaligenes genera, Pseudomonas genera etc. The bacteria to which it belongs are mentioned as a preferable example. From the viewpoint of safety and productivity, more preferably bacteria belonging to the genus Lalstonia, Capriavidas, Aeromonas, or Wautersia, still more preferably bacteria belonging to the genus Capriabidus or Aeromonas, still more preferably to the genus Capriabidus. It is a bacterium belonging to the genus, particularly preferably Capriavidus necator.

  When the prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing 3HH units is a recombinant prokaryotic microorganism strain into which a foreign PHA synthetase gene has been introduced by a genetic engineering method, the foreign PHA synthesis The enzyme gene is not particularly limited as long as it is a gene having a function of incorporating 3HH and producing a copolymerized PHA containing 3HH units. As such a PHA synthetase gene, for example, a PHA synthetase gene derived from Aeromonas caviae, which encodes an enzyme having the amino acid sequence set forth in SEQ ID NO: 1, or 85 relative to the amino acid sequence It includes, but is not limited to, a PHA synthetase gene having a% or more sequence identity and encoding a polypeptide having a 3HH unit-containing co-polymerized PHA synthesis activity. The above sequence identity is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more. Among these, a PHA synthetase gene capable of synthesizing P (3HB-co-3HH) as a 3HH unit-containing copolymer PHA is preferable, and in particular, a PHA synthetase having an amino acid sequence described in SEQ ID NO: 2 is encoded More preferred is the PHA synthetase gene.

  In the present invention, a recombinant prokaryotic microorganism strain obtained by introducing a PHA synthetase gene derived from Aeromonas caviae into Capriavidas necator is most preferable as the original prokaryotic microorganism.

  In the present invention, a gene encoding an enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity in the above-mentioned prokaryotic microorganism (hereinafter sometimes abbreviated as transgene) Introduce.

  The transgene is usually a gene which is not retained by prokaryotes but derived from eukaryotes. Thus, in the present invention, genes derived from eukaryotes are introduced into prokaryotic microorganisms. A transformant obtained by introducing the transgene into a prokaryotic microorganism having the PHA synthetase gene can produce a copolymerized PHA containing 3HH units at a higher composition ratio.

  Usually, the β-oxidation in prokaryotic microorganisms is a reaction from trans-2-enoyl-CoA via (S) -3-hydroxyacyl-CoA, and in this β-oxidation pathway, the monomer of the copolymerized PHA containing 3HH units is used. The R-shaped 3HH monomer is not formed. However, according to the present invention, (R) -3-hydroxyacyl-CoA can be produced from trans-2-enoyl-CoA in the prokaryotic microorganism by using the above-mentioned transgene as a carbon source by introducing the transgene into the prokaryotic microorganism. As a metabolic pathway is established, and this route leads to synthesis of (R) -3-hydroxyacyl-CoA having 6 carbon atoms, as a result, R-shaped 3HH monomer is generated, and the final compound It is presumed that the composition ratio of 3HH units in the copolymerized PHA increases.

  On the other hand, when 3HH unit-containing copolymerized PHA is to be produced in a culture using sugar as a carbon source, the reaction in the opposite direction to normal β-oxidation, from acetyl-CoA having 2 carbon atoms to 6 carbon atoms ( R) It may be necessary to synthesize -3-hydroxyacyl-CoA. Also in this case, by introducing the transgene into a prokaryotic microorganism, a metabolic pathway via (R) -3-hydroxyacyl-CoA is constructed, thereby efficiently producing R-shaped 3HH monomer. It is presumed that the compositional ratio of 3HH units in the copolymerization PHA of the final composition is increased.

  The transgene is not particularly limited as long as it is a gene encoding an enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity, but generally, the enzyme multifunctional enzyme type 2 The gene known as MFE2 gene which codes (MFE-2) is mentioned. Specific examples thereof include MFE2 gene (generally called MFE-1 gene) derived from Yarrowia lipolytica, MFE2 gene derived from Drosophila melanogaster etc., and MFE2 gene (generally called FOX2 gene) derived from Saccharomyces cerevisiae.

  Since the transgene is a gene derived from eukaryote, it may contain a non-translated region generally called intron. In such a case, the transgene in a state in which the base sequence corresponding to the intron has been removed may be introduced into a prokaryotic microorganism. In addition, codons in the transgene may be appropriately modified depending on the host.

The transgene is preferably the MFE2 gene encoding MFE-2. Specifically, for example, the MFE2 gene derived from Yarrowia lipolytica encoding the amino acid sequence represented by SEQ ID NO: 3, the MFE2 gene derived from Drosophila melanogaster encoding the amino acid sequence represented by SEQ ID NO: 4, or the like Genes which encode a polypeptide having at least 90% sequence identity to the amino acid sequence and which show trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity are mentioned. Be The above sequence identity is more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more. In Non-Patent Document 5, a PHA synthetase gene capable of incorporating a 3HH monomer was introduced. It is reported that P (3HB-co-3HH) can be produced using sugar as a carbon source by introducing crotonyl-CoA reductase (CCR) gene and ethylmalonyl-CoA decarboxylase (EMD) gene into Capriabidas necatol. It is done. Also in the transformant of the present invention, a CCR gene and / or an EMD gene may be introduced in addition to the transgene. By introducing the CCR gene and / or the EMD gene, in the case of using a sugar as a carbon source, the above-mentioned synthesis route of carbon number 6 (R) -3-hydroxyacyl-CoA is strengthened or streamlined and produced. The compositional ratio of 3HH units in the polymerized PHA is improved.

  In the present specification, CCR is an enzyme that reduces crotonyl-CoA having 4 carbons, which is an intermediate of fatty acid β-oxidation pathway, to produce butyryl-CoA. The origin of the CCR gene that can be used in the present invention is not particularly limited as long as the post-translational reductase has the above-mentioned crotonyl-CoA reductase activity. A gene encoding crotonyl-CoA reductase derived from C. cinnamonensis (GenBank Accession No. AF178673), a methanol-utilizing bacterium M. subtilis, and the like. A gene encoding crotonyl-CoA reductase derived from extorquens (NCBI-Gene ID: 7990208) and the like can be mentioned. Preferably, a gene encoding crotonyl-CoA reductase having the amino acid sequence set forth in SEQ ID NO: 5, or a poly having 90% or more sequence identity to the amino acid sequence and exhibiting crotonyl-CoA reductase activity It is a gene encoding a peptide.

  In the present specification, EMD means an enzyme which catalyzes the decarboxylation reaction of ethylmalonyl-CoA to butyryl-CoA produced in a side reaction with crotonyl-CoA reductase, propionyl-CoA carboxylase and the like. The origin of EMD is not particularly limited as long as it has this activity, and examples include mouse-derived ethylmalonyl-CoA decarboxylase having the amino acid sequence set forth in SEQ ID NO: 6. Examples of the gene base sequence encoding the amino acid sequence include, but not limited to, the base sequence described in SEQ ID NO: 7.

  If the prokaryotic microorganism into which the above transgene has been introduced has low or no glucose assimilation ability, the method of the present invention may be carried out by methods such as gene mutation, gene disruption, enhancement of gene expression, introduction of foreign genes, etc. The transformant can be imparted with or enhanced in glucose assimilation ability. For example, C.I. The necator H16 strain can not assimilate glucose because it does not have a glucose uptake gene. C. The method for imparting glucose assimilation ability to necator H16 strain is not particularly limited. For example, G which is the 793th base of nagE which is an incorporated gene of N-acetylglucosamine is replaced with C, and a transcription regulatory factor The method of giving glucose assimilation ability by destroying nagR which is the gene which codes the, (Journal of Bioscience and Bioengineering, vol. 113, 63 (2012)) is mentioned. Moreover, the method (Unexamined-Japanese-Patent No. 2009-225622) which provides glucose assimilation ability is also mentioned by introduce | transducing the foreign gene which codes a glucose transporter. Furthermore, in some cases, a method of introducing a glucose kinase gene is effective.

  In the transformant of the present invention, the introduced transgene or other gene may be present on a DNA such as a chromosome, a plasmid, a megaplasmid or the like possessed by a prokaryotic microorganism as a host, or on a plasmid vector or It may be present on DNA artificially incorporated into a transformant, such as on an artificial chromosome. However, from the viewpoint of retention of the introduced gene, it is preferably present on the chromosome or megaplasmid carried by the prokaryotic microorganism, and more preferably present on the chromosome carried by the prokaryotic microorganism. In addition, when a prokaryotic microorganism serving as a host originally retains these genes, gene expression can be achieved by substituting, deleting or adding a base sequence upstream of the originally retained gene instead of introduction. The amount may be increased.

  Methods for site-specific substitution or insertion of any DNA on DNA carried by a microorganism are well known to those skilled in the art, and can be used in producing the transformant of the present invention. Although not particularly limited, as a representative method, a method using transposon and the mechanism of homologous recombination (Ohman et al., J. Bacteriol., Vol. 162: p. 1068 (1985)), the mechanism of homologous recombination Method based on the site-specific integration that occurs and the dropout due to the second step of homologous recombination (Noti et al., Methods Enzymol., Vol. 154, p. 197 (1987)), coexistence of the sacB gene from Bacillus subtilis A method of easily isolating a strain of microorganism from which a gene has been eliminated by homologous recombination in the second step as a sucrose-supplemented medium-resistant strain (Schweizer, Mol. Microbiol., Vol. 6, p. 1195 (1992); Lenz Et al., J. Bacteriol., Vo. l.176, p. 4385 (1994)) and the like. Further, the method for introducing the vector into cells is not particularly limited, and examples thereof include a calcium chloride method, an electroporation method, a polyethylene glycol method, a spheroplast method and the like.

  For gene cloning and gene recombination techniques, see Sambrook, J. et al. et al. The techniques described in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989 or 2001), etc. can be used.

  The promoter for expressing the various genes described above is not particularly limited. It has a promoter of phaC1 gene of Capribidas necator, a promoter of phaP1 gene, lac promoter derived from E. coli, lacUV5 promoter, trc promoter, tic promoter, tac promoter, or a base sequence represented by artificially created SEQ ID NO: 8 Poe1 promoter etc. can be used.

2. Method for Producing PHA PHA can be produced by culturing the transformant of the present invention, and recovering the obtained PHA to produce PHA.

  In the production of PHA according to the present invention, the transformant is preferably cultured in a medium containing a carbon source, a nitrogen source which is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources.

  As a carbon source, any carbon source may be used as long as it can assimilate the transformant of the present invention, but preferably, saccharides such as glucose, fructose, sucrose, etc .; palm oil, palm kernel oil, corn oil, Oils and fats such as coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil and their fractionated oils; Fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid and mirinstic acid and derivatives thereof.

  Examples of the nitrogen source include ammonia; ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate; peptone, meat extract, yeast extract and the like. Examples of the inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like. Other organic nutrient sources include, for example, amino acids such as glycine, alanine, serine, threonine and proline; and vitamins such as vitamin B1, vitamin B12 and vitamin C.

  When culturing the transformant of the present invention, conditions such as culture temperature, culture time, pH at the time of culture, and the like are the host prokaryotic microorganisms such as, for example, Ralstonia, Capriabidas, Waurthia, Aeromonas, Escherichia, Alcaligenes. The conditions may be those commonly used in the culture of bacteria such as genus and pseudomonas.

  The type of PHA produced in the present invention is not particularly limited as long as it is a copolymerized PHA containing 3HH units, but 2-hydroxyalkanoic acid having 4 to 16 carbon atoms and 3-hydroxyalkanoic acid (except 3HH) And a copolymerized PHA obtained by polymerizing 3HH with one or more monomers selected from 4-hydroxyalkanoic acid, and more preferably a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid It is P (3HB-co-3HH). The type of PHA to be produced is, depending on the purpose, the type of PHA synthetase gene possessed or introduced separately by the prokaryotic microorganism to be used, the type of metabolic gene involved in the synthesis, culture conditions, etc. It can be selected as appropriate.

  In the present invention, after culturing the transformant, recovery of PHA from the cells is not particularly limited, but can be carried out, for example, by the following method. After completion of the culture, cells are separated from the culture solution by a centrifuge or the like, and the cells are washed with distilled water, methanol or the like, and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. The cell components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Furthermore, the supernatant is removed by filtration or centrifugation and dried to recover PHA.

  The analysis of the weight average molecular weight (Mw) of the obtained PHA and the monomer composition (mol%) such as 3HH can be performed by, for example, gel permeation chromatography, gas chromatography, nuclear magnetic resonance method or the like.

  EXAMPLES The present invention will be described in detail by way of examples, but the present invention is not limited by these examples. The entire genetic manipulation can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). In addition, enzymes used for gene manipulation, cloning hosts and the like can be purchased from market suppliers and used according to the description. The enzyme is not particularly limited as long as it can be used for genetic manipulation.

  The KNK005 strain used in the following Production Examples, Examples, and Comparative Examples is C.I. The transformant is a transformant in which a PHA synthetase gene derived from Aeromonas caviae (a gene encoding a PHA synthetase having the amino acid sequence described in SEQ ID NO: 2) is introduced onto the chromosome of necator H16 strain. In addition, KNK 005 Δ pha Z 1, 2, 6 is C .. It is a transformant in which the phaZ1,2,6 gene on the chromosome of necator H16 strain is deleted. These transformants can be prepared according to the methods described in US Pat. No. 7,384,766 and WO 2014/065253.

Preparation Example 1 Preparation of MFE2yl Expression Plasmid In this preparation example, a MFE2yl expression plasmid was prepared. The preparation was performed as follows.

  A DNA fragment (SEQ ID NO: 9) having the MFE2 (MFE2yl) gene sequence derived from Yarrowia lipolytica was obtained by PCR using synthetic oligo DNA. This DNA fragment is digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment is ligated with the plasmid vector pCUP2 described in WO2007 / 049716 digested with MunI and SpeI to obtain plasmid vector pCUP2-MFE2yl. I got

  Furthermore, a DNA fragment (SEQ ID NO: 10) having a trc promoter was obtained by PCR using a synthetic oligo DNA. This DNA fragment was digested with restriction enzymes EcoRI and MunI, and the obtained DNA fragment was ligated with the plasmid vector pCUP2-MFE2yl digested with MunI. From the obtained plasmid vector, a plasmid vector in which the trc promoter sequence was linked was selected in the direction in which the MFE2yl sequence was located downstream of the trc promoter sequence, and used as a plasmid vector pCUP2-trc-MFE2yl.

Example 1 Preparation of pCUP2-trc-MFE2yl / KNK005 Strain In this example, the plasmid vector pCUP2-trc-MFE2yl obtained in Production Example 1 was introduced into KNK005 strain to transform transformant pCUP2-trc-MFE2yl / KNK005 strain was obtained.

  The introduction of the plasmid vector into cells was carried out by electroinduction as follows. The gene transfer apparatus used Gene pulsar manufactured by Biorad, and the cuvette used gap 0.2 cm also manufactured by Biorad. In a cuvette, 400 μl of competent cells and 20 μl of expression vector were injected and set in a pulse device, and an electric pulse was applied under the conditions of electrostatic capacity 25 μF, voltage 1.5 kV and resistance value 800 Ω. After pulsing, shake the culture solution in the cuvette with Nutrient Broth medium (manufactured by DIFCO) at 30 ° C for 3 hours, and use the selection plate (NutrientAgar medium (manufactured by DIFCO), kanamycin 100 mg / L) at 30 ° C. The cells were cultured for 1 day to obtain the grown transformant pCUP2-trc-MFE2yl / KNK005 strain.

Production Example 2 KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6: Preparation of Poe1-ccr-emd Strain First, a plasmid for chromosome substitution was prepared. The preparation was performed as follows.

  A DNA fragment (SEQ ID NO: 11) containing a partial sequence of the nagE gene was obtained by PCR using synthetic oligo DNA. The resulting DNA fragment was digested with restriction enzyme SwaI. This DNA fragment was ligated with the vector pNS2X-sacB also described in JP 2007-259708 A similarly digested with SwaI using a DNA ligase (Ligation High (Toyobo Co., Ltd.)), and was upstream from the 793th base of the nagE structural gene A plasmid vector pNS2X-sacB + nagEG793C for chromosomal substitution containing a nucleotide sequence having a nucleotide sequence downstream of and a nucleotide sequence in which the 793th base of the nagE structural gene is substituted from G to C was prepared.

  Next, using the plasmid vector pNS2X-sacB + nagEG793C for chromosomal substitution, the chromosomal substitution strain KNK005ΔphaZ1,2,6 / nagEG793C was prepared as follows.

  E. coli strain S17-1 (ATCC 47055) is transformed with plasmid vector pNS2X-sacB + nagEG793C for chromosome substitution, and the resulting transformant is mixed and cultured on KNK005ΔphaZ1,2,6 strain and Nutrient Agar medium (Difco). Bonding transmission was performed.

  The resulting culture solution was collected on a Simmons agar medium containing 250 mg / L kanamycin (2 g / L sodium citrate, 5 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 1 g ammonium dihydrogenphosphate / l L. Seed on L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8), select the strain grown on the agar medium, and integrate the plasmid onto the chromosome of strain KNK005ΔphaZ1,2,6. Acquired stock. This strain was cultured for two generations in Nutrient Broth medium (manufactured by Difco), diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the grown strain was obtained as a strain from which the plasmid was removed. Furthermore, analysis with a DNA sequencer isolated a strain 1 strain in which G which is the 793rd base of the nagE structural gene on the chromosome was replaced by C. This mutated strain was designated as KNK005ΔphaZ1,2,6 / nagEG793C strain. The obtained KNK005ΔphaZ1,2,6 / nagEG793C strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. It is a strain in which a gene encoding a PHA synthetase having an amino acid sequence has been introduced, and G which is the 793rd base of the nagE structural gene has been substituted with C.

  Furthermore, a plasmid for gene disruption was prepared. The preparation was performed as follows.

  A DNA fragment (SEQ ID NO: 12) having a base sequence upstream and downstream of the nagR structural gene was obtained by PCR using a synthetic oligo DNA. The resulting DNA fragment was digested with restriction enzyme SwaI. This DNA fragment is ligated with the vector pNS2X-sacB also described in JP 2007-259708 A similarly digested with SwaI using a DNA ligase (Ligation High (made by Toyobo Co., Ltd.)), and the nucleotide sequences upstream and downstream from the nagR structural gene A plasmid vector for gene disruption pNS2X-sacB + nagRUD was prepared.

  Next, using the plasmid vector pNS2X-sacB + nagRUD for gene disruption, using the KNK005ΔphaZ1,2,6 / nagEG793C strain as a parent strain and using the same method as above, preparation of the gene disruption strain KNK005ΔphaZ1,2,6 / nagEG793C, dR strain is carried out went.

  The KNK005ΔphaZ1,2,6 / nagEG793C, dR strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. It is a strain in which a gene encoding a PHA synthetase having an amino acid sequence has been introduced, G which is the 793th base of the nagE structural gene has been substituted by C, and further deletion from the start codon to the stop codon of the nagR gene.

  Furthermore, a plasmid for chromosome transfer was prepared. The preparation was performed as follows. By PCR using synthetic oligo DNA, C.I. A DNA fragment (SEQ ID NO: 13) having a nucleotide sequence upstream and downstream of the phaZ2 gene of necator H16 strain was obtained. This DNA fragment was digested with restriction enzyme SwaI, and the vector pNS2X-sacB similarly described in JP-A-2007-259708 similarly digested with SwaI was ligated with DNA ligase (Ligation High (Toyobo Co., Ltd.)) to obtain pNS2X-sacB + Z2UDMS. Obtained. Next, a DNA fragment (SEQ ID NO: 14) having a Poe1 promoter, a CCR gene, and an EMD gene was obtained by PCR using artificial gene synthesis and synthetic oligo DNA. This DNA fragment is digested with restriction enzymes EcoRI and SpeI, and the obtained DNA fragment is ligated with pNS2X-sacB + Z2UDMS digested with MunI and SpeI and DNA ligase (Ligation High (Toyobo Co., Ltd.)) to obtain a plasmid for introducing chromosomes. The vector pNS2X-sacB + Z2 :: Poe1-ccr-emd was obtained.

  Furthermore, using the plasmid vector pNS2X-sacB + Z2 :: Poe1-ccr-emd for chromosome transfer, using the KNK005ΔphaZ1,2,6 / nagEG793C, dR strain as a parent strain and in the same manner as above, the gene-disrupted strain KNK005ΔphaZ1,2,6. Preparation of / nagEG793C, dR / Z2 :: Poe1-ccr-emd strain was performed.

  KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2 :: Poe 1-ccr-emd strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, and deletion from the start codon to the stop codon of the nagR gene is further performed, CCR gene and EMD gene were introduced into one copy each.

  Furthermore, C.I. A DNA fragment (SEQ ID NO: 15) having a nucleotide sequence upstream and downstream of the phaZ6 gene of necator H16 strain was obtained. This DNA fragment is digested with restriction enzyme SwaI, and the vector pNS2X-sacB also described in JP2007-259708 similarly digested with SwaI is ligated with DNA ligase (Ligation High (Toyobo Co., Ltd.)) to obtain vector pNS2X-sacB + Z6UDMS. I got Next, a DNA fragment (SEQ ID NO: 14) having the Poe1 promoter, CCR gene and EMD gene described above is digested with restriction enzymes EcoRI and SpeI, and the resulting DNA fragment is digested with MunI and SpeI, vector pNS2X-sacB + Z6UDMS The fragment was ligated with DNA ligase (Ligation High (Toyobo Co., Ltd.)) to obtain a plasmid vector pNS2X-sacB + Z6 :: Poe1-ccr-emd for introducing a chromosome.

  Furthermore, using the plasmid vector pNS2X-sacB + Z6 :: Poe1-ccr-emd for chromosome transfer, using the KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2 :: Poe1-ccr-emd strain as a parent strain and using the same method as above, The transgenic strain KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6 :: Poe1-ccr-emd strain was prepared.

  KNK 005 Δpha Z 1, 2, 6 / nag EG 793 C, dR / Z 2, Z 6: The Poe 1-ccr-emd strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, C is deleted from the start codon to the stop codon of the nagR gene, and further on the chromosome It is a strain into which two copies of the CCR gene and the EMD gene have been introduced.

(Example 2) pCUP2-trc-MFE2yl / KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6: Preparation of Poe1-ccr-emd strain The plasmid vector pCUP2-trc-MFE2yl prepared in Production Example 1 was carried out. KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6: described in Preparation Example 2 by introduction of electricity as described in Example 1, into the Poe1-ccr-emd strain, and pCUP2-trc-MFE2yl / KNK005ΔphaZ1,2, 6 / nag EG 793 C, dR / Z2, Z6 :: Poe 1-ccr-emd strain was obtained.

Production Example 3 Preparation of KNK143S / Z6 :: Poe1-ccr-emd Strain First, using the plasmid vector bAO / pBlu / SacB-Km for chromosome introduction described in JP-A-2013-9627, KNK005ΔphaZ1,2, Using the 6 / nag EG 793 C, dR strain as a parent strain, a promoter and SD sequence inserted strain ACP-bktB / Δpha Z1, 2, 6 / nag EG 793 C, d R strain was prepared by the method described in Production Example 2.

  The ACP-bktB / ΔphaZ1,2,6 / nag EG 793C, dR strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, C is deleted from the start codon to the stop codon of the nagR gene, and bktB (β A. just before the start codon of the ketothiolase gene. It is a strain into which a DNA consisting of a nucleotide sequence containing a promoter of a phaC gene of caviae and a ribosome binding sequence is inserted.

  Next, using the promoter described in WO 2015/115619 and the plasmid vector pNS2X-sacB + phaJ4bU-trc-phaJ4b for inserting an SD sequence, the ACP-bktB / ΔphaZ1,2,6 / nagEG793C, dR strain is used as a parent strain. A promoter and SD sequence inserted strain ACP-bktB / ΔphaZ1,2,6 / nagEG793C, dR / trc-J4b strain were prepared by the method described in Production Example 2.

  The ACP-bktB / ΔphaZ1,2,6 / nagEG793C, dR / trc-J4b strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, C is deleted from the start codon to the stop codon of the nagR gene, and the bktB gene is started Just before the codon A. It is a strain into which a DNA consisting of a nucleotide sequence comprising a promoter of phaC gene of caviae and a ribosome binding sequence is inserted, and a DNA consisting of a nucleotide sequence comprising a trc promoter and a ribosome binding sequence just before the start codon of phaJ4b gene.

  Furthermore, the method described in Production Example 2 using ACP-bktB / ΔphaZ1,2,6 / nagEG793C, dR / trc-J4b strain as a parent strain using chromosomal substitution vector pBlueASRU described in JP2008-29218A. KNK144S strain was produced.

  The KNK144S strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, C is deleted from the start codon to the stop codon of the nagR gene, and the bktB gene is started Just before the codon A. A DNA consisting of a base sequence containing the promoter and ribosome binding sequence of caviae phaC gene is inserted, and a DNA consisting of a base sequence comprising the trc promoter and ribosome binding sequence is inserted just before the start codon of phaJ4b gene, and further phaA structural gene sequence It is a strain in which a stop codon and a restriction enzyme NheI cleavage site are generated.

  Next, a promoter, a ribosome binding sequence and a plasmid for gene insertion were prepared. The preparation was performed as follows.

  By PCR using artificial gene synthesis and synthetic oligo DNA, a DNA fragment containing a ribosome binding sequence, a CCR gene, and an EMD gene, into which the base sequence shown in SEQ ID NO: 16 was introduced, was obtained. This DNA fragment was digested with restriction enzymes MunI and SpeI and ligated with the plasmid vector pNS2X-sacB + Z2UDMS digested with MunI and SpeI to obtain the plasmid vector pNS2X-sacB + Z2U-ccr-emd-Z2D.

  Next, a DNA fragment (SEQ ID NO: 17) having a trc promoter was obtained by PCR using a synthetic oligo DNA. This DNA fragment was digested with MunI and ligated with the plasmid vector pNS2X-sacB + Z2U-ccr-emd-Z2D digested with MunI described above. From among the obtained plasmids, a plasmid in which the trc promoter sequence is linked is selected by PCR in a direction in which ccr and emd are located downstream of the trc promoter sequence, and the promoter, ribosome binding sequence and plasmid vector for gene insertion pNS2X- sacB + Z2U-trc-ccr-emd-Z2D was obtained.

  Next, using a promoter, a ribosome binding sequence and a plasmid vector for gene insertion pNS2X-sacB + Z2U-trc-ccr-emd-Z2D, KNK143S strain was prepared by the method described in Production Example 2 using KNK144S strain as a parent strain.

  The KNK143S strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, C is deleted from the start codon to the stop codon of the nagR gene, and the bktB gene is started Just before the codon A. A DNA consisting of a nucleotide sequence containing the promoter and ribosome binding sequence of the caviae phaC gene is inserted, and a DNA consisting of the nucleotide sequence comprising the trc promoter and ribosome binding sequence is inserted immediately before the start codon of the phaJ4b gene, in the phaA structural gene sequence A stop codon and a restriction enzyme NheI cleavage site were generated, and the strain originally had the trc promoter, ribosome binding sequence, CCR gene and EMD gene inserted at the position where the phaZ2 gene was present.

  Furthermore, using plasmid vector pNS2X-sacB + Z6 :: Poe1-ccr-emd for chromosome transfer described in Preparation Example 2 and using the KNK143S strain as a parent strain and in the same manner as in Preparation Example 2, a transgenic strain KNK143S / Z6: Poe1-ccr-emd strain was produced.

  KNK143S / Z6 :: Poe1-ccr-emd strain is C.I. It is deleted from the start codon to the stop codon of phaZ6 gene and phaZ1 gene on the chromosome of necator H16 strain, and further deleted from the 16th codon to the stop codon of phaZ2 gene, and described in SEQ ID NO: 2 on the chromosome. A gene encoding a PHA synthetase having an amino acid sequence is introduced, G which is the 793th base of the nagE structural gene is replaced with C, C is deleted from the start codon to the stop codon of the nagR gene, and the bktB gene is started Just before the codon A. A DNA consisting of a nucleotide sequence containing the promoter and ribosome binding sequence of the caviae phaC gene is inserted, and a DNA consisting of the nucleotide sequence comprising the trc promoter and ribosome binding sequence is inserted immediately before the start codon of the phaJ4b gene, in the phaA structural gene sequence In this strain, a stop codon and a restriction enzyme NheI cleavage site are generated, and two copies of the CCR gene and the EMD gene are introduced on the chromosome.

(Example 3) pCUP2-trc-MFE2yl / KNK143S / Z6: Preparation of Poe1-ccr-emd strain The plasmid vector pCUP2-trc-MFE2yl prepared in Production Example 1 was produced by electro-introduction described in Example 1. The KNK143S / Z6 :: Poe1-ccr-emd strain described in Example 3 was introduced to obtain the pCUP2-trc-MFE2yl / KNK143S / Z6 :: Poe1-ccr-emd strain.

Comparative Example 1 PHA Production by Strain KNK005 The composition of the seed culture medium is 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / v% Na ₂ It was HPO ₄ · 12H ₂ O, 0.15 w / v% KH ₂ PO ₄ .

The composition of the production medium used PHA production _{1.1w / v% Na 2 HPO 4} · 12H 2 O, 0.19w / v% KH 2 PO 4, 0.13w / v% (NH 4) 2 SO 4, 0.1 w / v% MgSO ₄ · 7 H ₂ O, 0.1 v / v% trace metal salt solution (1.6 w / v% FeCl ₃ · 6 H ₂ O, 0.1 w / v% CaCl ₂ · 2 H in 0.1 N hydrochloric acid ₂ O, 0.02 w / v% CoCl ₂ · 6 H ₂ O, 0.016 w / v% CuSO ₄ · 5 H ₂ O, 0.012 w / v% NiCl ₂ · 6 H ₂ O dissolved. As a carbon source, palm kernel oil was added to the medium to be 1.5 w / v%.

  A glycerol stock (50 μL) of strain KNK005 was inoculated into a seed culture medium (5 mL), shake culture was performed at a culture temperature of 30 ° C. for 24 hours, and the obtained culture solution was used as a seed mother.

  PHA production culture inoculated the said seed mother 1.0 v / v% into the Sakaguchi flask which put 50 mL production culture medium, and culture | cultivated culture was performed at culture | cultivation temperature of 30 degreeC. After culturing for 72 hours, the cells were recovered by centrifugation, washed with methanol, freeze-dried, and the dry cell weight was measured.

  The PHA production amount was calculated as follows. 100 mL of chloroform was added per 1 g of the obtained dried cells, and stirred overnight at room temperature to extract PHA in the cells. After filtering off the cell residue, the solution was concentrated with an evaporator until the total volume became 1/3, hexane was gradually added in an amount of 3 times the volume of the concentrated solution, and left for 1 hour while being slowly stirred. The precipitated PHA was filtered off and vacuum dried at 50 ° C. for 3 hours. The weight of dry PHA was measured, and the PHA production amount was calculated. The results are shown in Table 1.

  The 3HH composition ratio of the produced PHA was measured by gas chromatography as follows. About 20 mg of dry PHA was mixed with 2 ml of sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform, sealed tightly, and heated at 100 ° C. for 140 minutes to obtain a methyl ester of PHA decomposition product. After cooling, 1.5 g of sodium hydrogencarbonate was added little by little to neutralize it, and the mixture was left until carbon dioxide gas evolution ceased. After 4 ml of diisopropyl ether was added and mixed well, it was centrifuged and the monomer unit composition of the PHA degradation product in the supernatant was analyzed by capillary gas chromatography. The gas chromatograph used Shimadzu GC-17A, the capillary column used GL Science's NEUTRA BOND-1 (column length 25 m, column internal diameter 0.25 mm, liquid film thickness 0.4 micrometer). He was used as a carrier gas, the column inlet pressure was 100 kPa, and 1 μl of the sample was injected. The temperature was raised at a rate of 8 ° C./min to the initial temperature 100 ° C. to 200 ° C., and further raised at a rate of 30 ° C./min to 200-290 ° C. As a result of analysis under the above conditions, the 3HH composition ratio of the obtained PHA is shown in Table 1.

  The PHA produced in this comparative example was P (3HB-co-3HH) containing 2.9 mol% of 3HH units.

Example 4 PHA Production by pCUP2-trc-MFE2yl / KNK005 Strain The composition of the seed culture medium was the same as that described in Comparative Example 1. When cultivating the plasmid vector-introduced strain in the seed culture medium, kanamycin was added to the seed culture medium to a final concentration of 100 μg / ml.

  The composition and carbon source of the production medium used for PHA production were the same as those described in Comparative Example 1.

  The pCUP2-trc-MFE2yl / KNK005 strain prepared in Example 1 was cultured in the same manner as in Comparative Example 1, and the PHA production amount and 3HH composition ratio were calculated in the same manner as in Comparative Example 1. The obtained PHA production amount and 3HH composition ratio are shown in Table 1.

  The PHA produced in this example was P (3HB-co-3HH) having a 3HH composition ratio of 6.3 mol%. That is, the introduction of the MFE2 gene produced a copolymerized PHA containing more 3HH units than the copolymerized PHA produced in Comparative Example 1.

Comparative Example 2 KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6: Production of PHA by Poe1-ccr-emd Strain The composition of the seed culture medium was the same as that described in Comparative Example 1. The composition of the production medium used for PHA production is the same as that described in Comparative Example 1, and 40 w / v% glucose aqueous solution is used as a single carbon source instead of palm kernel oil as a carbon source, and glucose is 2.0 w It was added to the production medium to be / v%.

  KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6 :: Poe1-ccr-emd strain prepared in Production Example 2 was cultured in the same manner as in Comparative Example 1, and PHA production amount and 3HH composition ratio were compared in Comparative Example 1 Calculated in the same manner as in. The obtained PHA production amount and 3HH composition ratio are shown in Table 1.

  The PHA produced in this comparative example did not contain 3HH units and was PHB.

Example 5 pCUP2-trc-MFE2yl / KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6: Production of PHA by Poe1-ccr-emd Strain The composition of the seed culture medium is the same as that described in Comparative Example 1. And When cultivating the plasmid vector-introduced strain in the seed culture medium, kanamycin was added to the seed culture medium to a final concentration of 100 μg / ml.

  The composition of the production medium used for PHA production is the same as that described in Comparative Example 1, and 40 w / v% glucose aqueous solution is used as a single carbon source instead of palm kernel oil as a carbon source, and glucose is 2.0 w It was added to the production medium to be / v%.

  The pCUP2-trc-MFE2yl / KNK005ΔphaZ1,2,6 / nagEG793C, dR / Z2, Z6 :: Poe 1-ccr-emd strain prepared in Example 2 was cultured in the same manner as in Comparative Example 1, and the PHA production amount and 3HH were obtained. The composition ratio was calculated in the same manner as in Comparative Example 1. The obtained PHA production amount and 3HH composition ratio are shown in Table 1.

  The PHA produced in this example was P (3HB-co-3HH) containing 1.4 mol% of 3HH units. In Comparative Example 2, a homopolymerized PHA not containing 3HH units was produced, whereas in this example, a copolymerized PHA containing 3HH units was produced by the introduction of the MFE2 gene.

Comparative Example 3 PHA Production by Poe 1-ccr-emd Strain KNK143 S / Z 6: The composition of the seed culture medium was the same as that described in Comparative Example 1. The composition of the production medium used for PHA production is the same as that described in Comparative Example 1, and 40 w / v% glucose aqueous solution is used as a single carbon source instead of palm kernel oil as a carbon source, and glucose is 2.0 w It was added to the production medium to be / v%.

  The KNK143S / Z6 :: Poe1-ccr-emd strain prepared in Production Example 3 was cultured in the same manner as in Comparative Example 1, and the PHA production amount and 3HH composition ratio were calculated in the same manner as in Comparative Example 1. The obtained PHA production amount and 3HH composition ratio are shown in Table 1.

  The PHA produced in this comparative example was P (3HB-co-3HH) containing 1.1 mol% of 3HH units.

Example 6 pCUP2-trc-MFE2yl / KNK143S / Z6: PHA Production by Poe1-ccr-emd Strain The composition of the seed culture medium was the same as that described in Comparative Example 1. The composition of the production medium used for PHA production is the same as that described in Comparative Example 1, and 40 w / v% glucose aqueous solution is used as a single carbon source instead of palm kernel oil as a carbon source, and glucose is 2.0 w It was added to the production medium to be / v%.

  The pCUP2-trc-MFE2yl / KNK143S / Z6 :: Poe1-ccr-emd strain prepared in Example 3 was cultured in the same manner as in Comparative Example 1, and the PHA production amount and the 3HH composition ratio were the same as in Comparative Example 1. Calculated by. The obtained PHA production amount and 3HH composition ratio are shown in Table 1.

  The PHA produced in this example was P (3HB-co-3HH) containing 4.7 mol% of 3HH units. That is, the introduction of the MFE2 gene produced a copolymerized PHA containing more 3HH units than the copolymerized PHA produced in Comparative Example 3.

Claims (10)

  For a prokaryotic microorganism having a PHA synthetase gene capable of synthesizing a copolymerized PHA containing 3HH units, an enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity is encoded A transformant producing a co-polymerized PHA containing 3HH units, into which a gene for   The transformant according to claim 1, further comprising a gene encoding crotonyl-CoA reductase (CCR).   The transformant according to claim 2, further comprising a gene encoding ethylmalonyl-CoA decarboxylase.   The gene encoding the enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity is a gene derived from Yarrowia lipolytica. The transformant as described in-.   The gene encoding the enzyme having trans-2-enoyl-CoA hydratase activity and (R) -3-hydroxyacyl-CoA dehydrogenase activity is a gene derived from Drosophila melanogaster. The transformant as described in a term.   The transformant according to any one of claims 1 to 5, wherein the prokaryotic microorganism is a bacterium.   The transformant according to any one of claims 1 to 6, wherein the prokaryotic microorganism is a bacterium belonging to the genus Capriavidus.   The transformant according to any one of claims 1 to 7, wherein the prokaryote is Capriavidas necator.   A method for producing a copolymerized PHA containing 3HH units, comprising the step of culturing the transformant according to any one of claims 1 to 8.   The method according to claim 9, wherein the copolymerization PHA is P (3HB-co-3HH).